The present invention relates to compounds and compositions for use in the treatment of diabetes or other hyperglycaemic defects of carbohydrate metabolism, methods of making the compositions and methods of treating diabetes or other hyperglycaemic defects in carbohydrate metabolism by administration of said compounds or compositions.
Diabetes mellitus is a metabolic disorder in which the ability to oxidise carbohydrates is reduced or completely lost, resulting in hyperglycaemia (raised blood sugar), polyuria (increased output of urine) and glucosuria (appearance of sugars (e.g. dextrose) in the urine). Diabetes has been recognised as a major disease for centuries. In addition to defective carbohydrate metabolism, it can also lead to altered metabolism of lipids and proteins and patients are at risk of complications from vascular disease which are always serious and may be fatal.
Diabetes results from failure of the Islets of Langerhans (xcex2) cells of the pancreas to produce sufficient insulin. It can also arise as a result of auto-immunity directed against Islet tissue and altered efficiency of insulin receptors. Temporary hyperglycaemia, which may not be sufficiently severe to be classified as diabetes, may occur due to hormonal imbalance during pregnancy.
Inositol (also known as meso-inositol, myo-inositol and cyclohexitol) is a growth factor and vitamin of the B complex which is widely distributed in plants and animals. Inositol appears to be involved physiologically in lipid metabolism and has been proposed as a second messenger for insulin.
The incidence of diabetes varies geographically and this is summarised in Table 1.
It is estimated that there is an equal number of undiagnosed cases. The morbidity and mortality associated with diabetes mellitus have prompted the search for effective treatments.
Current classification of diabetes mellitus (DM) distinguishes several types of disease and these have a bearing on treatment. Insulin-dependent diabetes mellitus (IDDM or Type I) is characterised by xcex2-cell antibodies and zero endogenous insulin levels; these patients are dependent on exogenous insulin to prevent ketoacidosis and death, and respond only to insulin or insulinomimetics. Noninsulin-dependent diabetes mellitus (NIDDM or Type II) refers to a lack of xcex2-cell antibodies, continued presence of endogenous insulin, and insulin resistance; these patients may or may not use insulin for symptom control but do not need it for survival. The symptoms of this type would be ameliorated by hypoglycaemics and by antagonists of glucagen. This group has been further sub-divided into obese and non-obese NIDDM. Other, hormonally induced forms of DM or DM due to pancreatic tissue damage are recognised. There is a spectrum of severity of diseases, for example xe2x80x9cimpaired glucose tolerancexe2x80x9d is present when individuals have plasma glucose levels intermediate between normal and those considered to be diabetic.
The disease has been treated, across the spectrum of severity, with pharmaceutical preparations, although there are disadvantages attaching to each of the major types of therapeutic agent. This prompts the search for new drug treatments, particularly those that are active when taken orally.
The main types of drug treatment currently available are:
Insulin, obtained form animal pancreas or produced in genetically modified micro-organisms, is available in a variety of forms for parenteral use. Insulin is destroyed in the gastro-intestinal tract and is almost invariably given by injection.
The need for treatments which do not need to be given by injection has long-been recognised, and several types of anti-diabetic agents have been introduced. Medicines based on hypoglycaemic, anti-hyperglycaemic agents, xcex1-glucosidase inhibitors, and hydrocolloid substances are licensed by regulatory authorities for the treatment of diabetes mellitus. The main groups of antidiabetic agents include, but are not limited to, the following:
Sulphonylureas, for example gliclazide, are a group of agents which cause hypoglycaemia by stimulating insulin release from pancreatic xcex2-cells.
Biguanides, exemplified by metformin, do not stimulate release of insulin but act by increasing the sensitivity of peripheral tissues to insulin.
xcex1-glucosidase inhibitors such as acarbose reduce intestinal absorption of carbohydrates and blunt the rise in plasma glucose which occurs after meals in both normal and diabetic subjects.
Several plant-based products, for example guar gum and ispaghula, contain colloidal substances which produce a bulky hydrated gel (soluble fibre) in the gastro-intestinal tract. The activity of this type of compound is essentially mechanical; when given with food the hydrated fibre entraps sugar and other carbohydrate molecules and slows down the absorption of carbohydrate from food. It thus blunts the postprandial rise in plasma glucose. Licensed products based on these actions are used as adjunctive therapy in the treatment of diabetes.
Each of the groups of orally active agents have limitations on efficacy and may also produce side effects. There is therefore a need for additional oral anti-diabetic agents which are effective and safe.
Some plant species have been claimed to contain antidiabetic constituents. A comprehensive review of plants which have been traditionally used for the treatment of diabetes has been published by Day (Phd thesis, Kings College School of Medicine and Dentistry, 1987) who also evaluated a number of these herbs including Allium cepa, Momordica charantia, and Pterocarpus marsupium, 
Artemisia judaica is used in Libyan traditional medicine as an infusion for the treatment of xe2x80x9cwasting diseasexe2x80x9d, almost certain diahetes mellitus. Little scientific work has been done on this herb although various Artemisia species are known to have some pharmacological activity (see Table 2).
Artemisia herba-alba, a closely related herb from Iraq has been found have an anti-diabetic effect. (Al-Waili, 1986, 1988; Twaij and Al-Badr, 1988). Extracts of Artemisia judaica have been found to contain some active pharmacological agents (Gallal et al, 1974, Abdalla and Zarga, 1987). Subramoniam et al (J Ethno-pharmacology 50 (1996) 13-17) have investigated the effects of A Pallens on normal and alloxon-induced diabetic rats. They used aqueous methanol and acetone extracts of the plant.
The use of all the above plants is limited by their toxicity, especially their mutagenicity. Toxicity data are essential for determination of the balance of risk and benefit and to determine the utility of therapeutic treatments.
We have found that crude extracts, such as infusions, prepared from Artemisia app, especially A judaica, have a positive mutagenic effect when tested in a conventional Ames type bacterial mutagenicity test. This mutagenicity is sufficient to preclude the development of the extracts as a commercial pharmaceutical product. When considering candidates for a potential pharmaceuticals, the therapeutic index (ratio of efficacy to toxicity) must be as high as possible. The present inventors have shown an extract to have activity in a chemically-induced rat model of Type 1 diabetes mellitus and in in vitro tests using isolated hepatocytes (liver cells). Surprisingly, they have been able to separate the efficacy and the mutagenicity to isolate a fraction from an Artemisia herb which has a significantly reduced mutagenic activity, whilst retaining the efficacy of the herb.
The present invention thus provides a purified extract of a plant from the Artemisia family which retains pharmaceutical efficacy while having lowered mutagenicity.
The invention also provides a pharmaceutical which is at least one composition of the invention, optionally in combination with a pharmacologically acceptable exipient. The pharmaceutical compositions of the invention may be suitable for oral or parenteral administration. The present invention further provides a method of a making pharmaceutical composition comprising mixing an extract, composition or compound of the invention with a pharmaceutically acceptable exipient.
The present invention also contemplates a method of treating hyperglycaemia, diabetes or other hyperglycaemic disorders of carbohydrate metabolism by administration of an extract or pharmaceutical composition of the invention. Also provided are methods of purifying an extract or compound according to the invention and the use of such extracts or compounds in the preparation of a medicament for the treatment of hyperglycaemia, diabetes or other hyperglycaemic defects of carbohydrate metabolism.
In the present invention a method is described of fractionating the extract to improve its utility by reducing its toxicity; while retaining its essential therapeutical benefit. By further fractionation it is possible to make extracts which have insulin-like activity and which have glucagon antagonist activity. Both of these activities are useful in a diabetes treatment for humans and animals. Using this method, it has been possible to produce extracts which have the required balance of insulin-stimulating and glucagon-antagonising actions to provide a treatment for DM. Surprisingly it has been found that both of these activities are desirable for optimum anti-diabetic activity in animals and humans.
Species related to Artemisia judaica are known to have been used in other ethnic cultures for similar purposes, but the toxicity attending such use is unacceptable. The data provided here show that gradient extraction of a plant extract yields fractions with anti-diabetic activity which retain the clinically useful action and have reduced toxicity.
Additional experimental work has provided further elucidation of fractions obtained from a concentrated aqueous extract of Artemisia spp. Surprisingly, it proved possible by using other chromatographic mobile phases; graded by altering the concentration of at least one component, to further fractionate. This reveals two or more fractions; one of which is flavonoid, glycoside- and polysaccharide-rich, and the other a terpenoid- and triterpenoid-rich fraction. Both have been tested for isulinomimetic and glucagon antagonist activity. The fraction which contains predominantly flavonoids (including glycosides) is insulinomimetic and is not eluted when the column is washed with water. This allows unabsorbed water-soluble material which is inactive in the GPa test to be removed. When the concentration of methanol in the eluting solvent (mobile phase) is increased, preferably in a stepwise fashion, most of the flavonoids are eluted at concentrations up to 40% methanol v/v; none were detected in the 70% fraction. Fractions eluted at methanol concentrations between 60% and 70% alcohol had optimal glucagon antagonist activity. By the use of a stepwise increase in alcohol concentration in the mobile phase, it is possible to effect a separation of fractions having respectively predominantly insulin-like and glucagon antagonist activities. It is thought that the 20-50% fraction contains triterpenoid components.
A method for preparing an anti-hyperglycaemic or anti-diabetic compound or extract according to the invention may comprise the steps of extraction of Artemisia judaica, Artemisia herba-alba or another herb of the Artemisia family with water, concentration to give a dry extract, washing with alcohol, preferably a lower aliphatic alcohol such as ethanol, chromatographic separation on e.g. a silica gel or an ion exhange column, elution with buffer and, optionally, processed further by concentration of the resulting solution or by drying. Desirably 100 g of Artemisia herb are extracted with water to give 12-20 g of dried extract.
The fractions eluted from the chromatography column may be analysed for anti-hyperglycaemic activity for example in an in vitro test such as those discussed herein and the active fractions combined for further processing, or processed separately. Alternatively, once the active fraction(s) have been identified, repetitive or batch processes may be employed to extract selectively or only those fractions from the plant. Drying may be by evaporation at elevated temperature and reduced pressure, e.g. at 30-45xc2x0 C., preferably 35-40xc2x0 C. or at 50xc2x0 C.
In accordance with the present invention, compositions provided may be administered to individuals. Administration is preferably in a xe2x80x9ctherapeutically effective amountxe2x80x9d, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially. For example, the present invention may provide a pharmaceutical composition for use as an adjunct to insulin therapy.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable exipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or parenteral e.g. by injection (cutaneous, subcutaneous or intravenous), or by administration to the buccal or nasal mucosae.
Formulations may be such as to provide a sustained release preparation for oral or parenteral use, such as by trans-dermal administration.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Insulin can have a direct effect on the liver, for example can influence steroid metabolism in isolated rat hepatocytes. By investigating the effect of the Artemisia extract on isolated hepatocytes, one can compare the biological effects of insulin and the extract. In the work leading to the present invention, the in vivo anti-diabetic activity of Artemisia judaica extracts has been tested in STZ diabetic and control rats, and these in vivo investigations complement hepatic Gpa activity tests in isolated hepatocytes.
Further details and aspects of the present invention will be apparent from the following examples and Figure which are presented by way of illustration and not limitation.